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Also called the active roster, the 25-man is composed of 25 players who are playing for their Major League team. They are the starting eight (NL) or nine (AL) position players, pitchers, and reserve players on the team. Players on the active roster are also on the 40-man roster. These players are generally the only ones who dress in uniform and are the only ones who may take the field in a game at any time. Typically, only players on the 25-man roster, and players who are on the disabled list but were on the 25-man roster, travel on road trips with the Major League team.

Typically, a 25-man roster will consist of five starting pitchers, seven relief pitchers, two catchers, six infielders, and five outfielders. In the American League a full-time designated hitter is usually classified as either an infielder or an outfielder, not a DH, because most DHs do play first base, left field, or right field from time to time.

Since the 2012 season, teams have been allowed 26-man rosters for "day-night" doubleheaders—two games scheduled on the same day, but with the stadium cleared between games, and separate tickets sold for each game—as long as the doubleheaders have been scheduled with at least 48 hours notice.[1]

Also called the expanded roster, the 40-man is composed of all the players in a Major League club's organization who are signed to a major-league contract. These are the players who are able to be called up to the 25-man roster at any given time. Also on the 40-man are any players on the 15-day disabled list and minor league players who are signed to a major-league contract but are on an "optional assignment" to the minors. (Each player has three "option years" to be sent to the minors once on the 40-man before they must be placed on waivers to be sent there.) Players who were on the 40-man but are placed on the 60-day disabled list are taken off the 40-man until the time on the DL is over. The same applies to players who are suspended. Because players on the 60-day DL are taken off the 40-man with no risk of losing the player, MLB teams often transfer injured players from the 15-day DL to the 60-day DL so that they can add another player to the 40-man without having to designate a player for assignment. Designating for assignment is the removal of a player from the 40-man, whereby the team has 10 days to trade the player, release him, or send him to the minors.

On September 1, the Major League team's roster expands from the 25-man active roster to the entire 40-man roster. At this point, any player on the 40-man roster can play for the Major League team. September call-ups are players from the minors who are playing in September to get Major League experience and, especially for teams in contention, to provide reinforcements down the stretch.

A postseason roster takes effect only if a team clinches a playoff berth. Players who are part of the team's final roster at the end of the regular season are eligible to participate in the postseason. Any player who has been traded from a different team, spent time in the Minor Leagues, or signed later in the season with the team (no later than August 31) are eligible to participate in the postseason. A postseason roster is allowed up to 25 active players. Other players who are not on the 25-man active roster will be placed on the secondary squad. Players who are on the disabled list or any other non-active transaction by the end of the regular season will have their transactions passed on in the postseason. Rosters for a series are set at the beginning of the series and no changes to the 25-man active roster are allowed except when a player is moved to the disabled list or any other inactive transaction. If a player is moved to the disabled list or another inactive transaction during a series, it then becomes ineligible to be returned to the 25-man active roster for the remainder of the series as well as the next series if applicable. If any player goes on any inactive transaction, any player from the secondary squad can be promoted to the 25-man active roster for the remainder of the series if applicable.[2]

To be eligible for the postseason active roster, a player must have either been on that team's active roster or disabled list as of midnight ET on August 31 of that year and not placed on the 60 day disabled list after August 1.[3] The one exception is for replacing players on the disabled list. Any injured player who is eligible for postseason play may be replaced by any player that was on an active or disabled list for either that team or any of its affiliated minor league teams at midnight August 31.[4] Players who do not participate in one game in the Majors before September 1 with the team's organization of the regular season will be declared ineligible for the team's 25-man active roster of the postseason and must be placed on either the restricted list or the secondary squad.

All other professional players affiliated with Major League Baseball are signed to minor-league contracts. They can receive an invitation to spring training with their organization's Major League team without being on the 40-man roster. Two types of players generally receive a non-roster invitation: prospect players who are there to gain experience and face tougher competition as well as receive instruction from the Major League team's coaching staff; and veteran players who were not offered any major league contract by a club. The veteran player is usually signed to a "two-way" salary option—one for their time in the minors and another if they are placed on the 40-man during the season. All spring training invitees are under some sort of contract, to avoid liability if an injury were to occur to the player.